Patent Application
20070286810
The technology described herein relates to particles linked covalently to luminescent lanthanide(III) chelates. The particles are prepared by polymerizing lanthanide(III) chelate derivatives in the presence of monomers. Because the resultant particles are covalently bound to the lanthanide(III) chelates, the signal obtained generally does not decrease as the function of time due to leaking upon storage. Because the lanthanide(III) chelates are luminescent, no additives that promote luminescence, such as phosphine oxides, are needed in the polymerization matrix. In addition, because the beads can be made using organic polymers or copolymers, such as polystyrene, they can be stable under basic conditions.
It has been shown previously that lanthanide (III) chelate detection sensitivity can be enhanced by incorporating chelates into particles. Beads containing lanthanide(III) chelates have most commonly been prepared simply by swelling the chelates into the polymer, as is described, for example, in Cummins, C. M., Koivunen, M. E., Stephanian, A., Gee, S. J., Hammock, B. D., Kennedy, I. M., 2006, Biocencors and Bioelectronics, 21, 1077. In addition, silica based nanobeads have been described by Hai, X., Tan, M., Wang, G., Ye, Z., Yuan, J., Matsumoto, K., 2004, Anal. Sci., 20, 245 and Sun, X., Wuest, M., Kovacs, Z., Sherry, A. D., Motekaitis, R., Wang, Z., Martell, A. E., Welch, M. J., Anderson, C. J., 2003, J. Biol. Inorg. Chem., 8, 217. Particles produced using nanodispersions of particle sizes in the middle or lower nanometer range (50-500 nm) are described, for example, in Horn, D., Rieger, J., 2001, Angew. Chem. Int. Ed. Engl., 40, 4330.
The invention provides a method for preparing particles comprising a lanthanide chelate. The method involves polymerizing a lanthanide chelate derivative with one or more monomers, wherein the lanthanide chelate derivative has the formula M-L-Y (I), wherein Y is a lanthanide chelate; L is a linker and M is a polymerizable moiety.
In an embodiment, the polymerizable moiety M in the lanthanide chelate derivative of formula (I) is a pendent vinyl group, acrylate group, methacrylate group, ethacrylate group, 2-phenylacrylate group, vinylketone group, acrylamide group, methacrylamide group, itaconate group or a styrene group. Specific examples include methacrylamide or acrylamide.
The compound of formula (I) can be allowed to polymerize with one or more organic monomers. Examples of organic monomers include styrene, vinyl alcohol, acrylic acid, metacrylic acid and esters and amides derived thereof. A specific example monomer is styrene.
The lanthanide chelate can be a luminescent lanthanide chelate. In an embodiment, the lanthanide chelate derivative is typically made of a chromophoric moiety comprising one or more aromatic units, and a chelating part. Exemplary compounds include those of shown below:
wherein
R is independently furyl, thiophenyl, or trialkoxyphenyl;
n is independently 1 or 2; and
L is a linker; and
Ln is europium, terbium, samarium, dysprosium or gadolinium; and
M is a polymerizable moiety.
The linker -L- is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkyl containing 1-12 carbon atoms, ethynediyl(—C≡C—), ethylenediyl(—C═C—); ether (—O—), thioether (—S—), amide (—CO—NH— and —NH—CO— and —CO—NR′ and —NR′—CO—), carbonyl(—CO—), ester (—COO— and —OOC—), disulfide (—SS—), diaza, (—N═N—), thiourea (—NH—CS—NH—) or a tertiary amine (—NR′—), where R′ represents an alkyl containing less than 5 carbon atoms.
The polymerization can be performed in the presence of a cross-linking agent such as divinylbenzene.
The size of the particle is generally below 500 nm, such as below 300 nm. In an embodiment, the diameter of the particle is in the range 10 . . . 150 nm, such as 90 . . . 120 nm.
The organic polymer or copolymer of the particle can be constructed, for example, of from monomers of vinyl, acrylate, methacrylate, ethacrylate, 2-phenylacrylate, vinylketone, vinyl alcohol, acrylamide, methacrylamide, itaconate or styrene.
The technology described herein is further elucidated by the following non-restricting examples. The structures and synthetic routes employed in the experimental part are depicted in Schemes 1 and 2. Experimental details are given in Examples 1-9. Scheme 3 describes a schematic preparation of a polymerizable terbium chelate. Properties of the nanospheres prepared are collected in Table 1.
Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck). All dry solvents were from Merck and they were used as received. NMR spectra were recorded on a Brucker 250 on a Jeol LA 400 spectrometer operating at 250.13 and 399.7 MHz for 1H, respectively. The signal of TMS was used as an internal reference. Coupling constants are given in Hertz. ESI-TOF mass spectra were recorded on an Applied Biosystems Mariner instrument. Luminescence measurements were measured with a PerkinElmer LS-5 luminescence spectrometer. IR and UV-spectra spectra were recorded on a PerkinElmer Spectrum One and Shimatzu 2400 instruments, respectively. Particle size analyses were performed on a Coulter LS-230 instrument, and are based on volume statistics. Photophysical properties of the nanoparticles prepared were measured as disclosed in Latva, M., Takalo, H., Mukkala, V.-M., Matachescu, C., Rodriques-Ubis, J. C., Kankare, J., 1997, J. Lumin., 35, 149 but the chelate concentration measurements were based on the weight of dry beads.
3-Aminoacetophenone (20.9 g, 0.15 mol) was dissolved in dry pyridine (60 mL) on an ice-water bath. Methacryloyl chloride (26.1 mL, 0.24 mol) was added dropwise during ½ h, and the mixture was stirred for an additional ½ h. The stirring was continued for 3 h at RT. All volatiles were removed in vacuo. The residue was dissolved in dichloromethane (150 mL), washed with 0.5 M HCl (2·100 mL) and water (2·100 mL) and dried (Na2SO4). Purification was performed on silica gel. The column was first eluted with CH2Cl2 to elute fast migrating impurities, and then with 5% (v/v) methanolic dichloromethane to elute the product. Yield was 18.1 g (59%). 1H NMR (CDCl3): 8.10 (1H, m); 8.00 (1H, br); 7.95 (1H, m); 7.43 (1H, m); 7.23 (1H, m); 5.85 (1H, s); 5.51 (1H, s); 2.61 (3H, s); 2.08 (3H, s). ESI-TOF MS: required for C12H14NO2+ 204.10 (M+H),+ found 204.08.
To a strirred solution of compound 1 (11.9 g, 58.55 mmol) in dry THF (100 mL) was added portionwise sodium hydride (3.52 g, 88 mmol; 60% dispersion in oil). After 5 min, ethyl trifluoroacetate (13.9 mL, 0.18 mol) was added, and the mixture was strirred for an additional 1 h before being concentrated in vacuo. The residue was suspended in ethyl acetate (220 mL) and acidified with 10% aqueous H2SO4 (80 mL), and washed with water. The organic layer was separated and dried over Na2SO4. Purification on silica gel (eluent, petroleum ether, bp. 40-60° C./ethyl acetate, 1:1, v/v) yielded 8.71 g (50%) of the title compound. 1H NMR (CDCl3): 8.14 (1H, s); 7.87 (1H, d, J 7.5); 7.74 (1H, br s); 7.74 (1H, d, J 7.8); 7.48 (1H, t, J 8.0); 6.58 (1H, s); 5.85 (1H, s); 5.54 (1H, s); 2.09 (3H, s). λmax(EtOH)/nm: 208, 250, 326. ESI-TOF MS: required for C13H13F3NNaO3+ 322.07 (M+Na),+ found 322.03.
Compound 2 (9.8 g, 32.7 mmol) was dissolved in the mixture of abs. ethanol (100 mL) and piperidine (3.2 mL), and the mixture was warmed to 45° C. Europium chloride hexahydrate (2.40 g, 6.54 mmol, predissolved in 20 mL of water) was added dropwise. The mixture was allowed to cool to RT, and water (100 mL) was added dropwise. The precipitation formed was collected by filtration, washed with water, and dried in vacuo. Yield was 7.9 g. λmax(H2O+1% DMF, v/v)/nm: 326 (ε 50552). ν/max(KBr) cm−1: 3440, 1663, 1620, 1586, 1534, 1489, 1301, 1187, 1138, 780, 580. Exmax 614 nm; Emmax 353 nm (tris-saline buffer, pH 7.75).
4-Bromo-6-bromomethyl-2-pyridylmethylenenitrilobis(acetic acid) di(tert-butyl ester) (4, 8.50 g, 16.7 mmol) and 6-tert-butoxycarbamoylhexane-1,6-diamine (1.80 g, 8.4 mmol) were dissolved in dry acetonitrile (60 mL). K2CO3 (9.2 g, 66.8 mmol) was added, and the mixture was heated overnight at 50° C. The precipitation formed was removed by filtration, and the filtrate was concentrated. Purification on silica gel (eluent: petroleum ether, bp 40-60° C.: ethyl acetate, from 10:1 to 5:2, v/v) yielded 5.9 g (65%) of compound 5. 1H NMR (CDCl3): δ 7.73 (2H, d, J 1.9); 7.58 (2H, d, J 1.9); 4.00 (4H, s); 3.74 (4H, s); 3.47 (8H, s); 3.08 (2H, q, J 5.5); 2.51 (2H, t, J 7.2); 1.46 (36H, s); 1.44 (9H, s); 1.53-1.42 (4H, m); 1.33-1.22 (4H, m). ν/max(film) cm−1: 3401 (N—H); 1734 (C═O); 1565 (arom. C—C). λmax(EtOH)/nm 268. ESI-TOF MS for C49H78Br2N6O10 (M+2H)2+: calcd, 536.22; found, 536.18.
Compound 5 (2.85 g, 2.66 mmol) and 2-(tributylstannyl)-thiophene (1.86 mL, 5.86 mmol) were dissolved in dry DMF (25 mL) and deaerated with argon. (Ph3P)4Pd (0.215 g, 0.22 mmol) was added, and the mixture was stirred at 90° C. for 6 h in dark. The mixture was cooled to room temperature and concentrated in vacuo. Purification was performed on silica gel (eluent: petroleum ether, bp 40-60° C.: ethyl acetate: triethylamine, from 5:1:1 to 5:3:1, v/v/v). Yield was 2.2 g (76%). 1H NMR (CDCl3): δ 7.76 (2H, s); 7.70 (2H, s); 7.55 (2H, d J 3.1); 7.36 (2H, d, J 4.9); 7.09 (2H, m); 4.05 (4H, s); 3.82 (4H, s); 3.50 (8H, s); 3.03 (2H, m); 2.60 (2H, m); 1.49-1.43 (4H, m); 1.45 (36H, s); 1.42 (9H, s); 1.39-1.32 (4H, m). νmax (film)/cm−1 1730 (C═O). λmax(EtOH)/nm 293. ESI-TOF-MS for C57H85N6O10S2(M+2H)2+: calcd, 539.29; obsd, 539.23.
Compound 6 (2.16 g, 2.00 mmol) was dissolved in trifluoroacetic acid (25 mL), and the mixture was stirred for 2 h at room temperature before being concentrated. The residue was triturated with diethyl ether. The precipitation formed was filtered, washed with diethyl ether and dried in vacuo. Yield was quantitative. 1H NMR (DMSO-d6): 7.90 (2H, s); 7.78 (2H, d, J 4.9); 7.73 (2H, d, J 3.4); 7.71 (2H, s); 7.25 (2H, dd, J 3.4 and 4.9); 3.95 (4H, s); 3.30 (8H, s); 3.22 (2H, m); 2.74 (2H, m); 1.78 (2H, m); 1.50 (2H, m); 1.33-1.23 (4H, m); νmax(KBr)/cm−1 1735, 1675, 1609 (C═O); 1559 (arom. C—C). λmax(EtOH)/nm 300.
Compound 7 (1.9 g) was dissolved in water (30 mL), and pH of the solution was adjusted to 6.5 with solid NaHCO3. Europium chloride hexahydrate (0.81 g, 2.2 mmol; predissolved in 30 mL of water) was added dropwice keepimg pH at ca 6. The mixture was stirred for 1.5 h at RT. pH was rised to 8.5 with aq. NaOH. The precipitation was removed by centrifugation. The clear solution was collected and concentrated in vacuo. It was used for the next step without further purification. νmax(KBr)/cm−1 1684, 1638, 1615 (C═O); 1552 (arom. C—C). λmax(EtOH)/nm 312. ESI-TOF-MS for C36H40EuN6O8S2−(M−H)−: calcd, 901.16; obsd, 901.16.
Compound 8 (2.6 g, 2.8 mmol) was dissolved in the mixture of water (20 mL), THF (40 mL) and DIPEA (1.7 mL). Methacroyl chloride (0.42 g, 4.0 mmol) was added, and the mixture was stirred for 5 min at RT before being concentrated in vacuo. The residue was suspended in chloform (30 mL). The precipitation formed was removed by filtration. The filtrate was concentrated to give the title compound. ESI-TOF-MS for C40H44EuN6O9S2− (M−H)−: calcd, 969.18; obsd, 969.18. The partition coefficient of compound 9 between H2O and CHCl3, was ca 1:1.
A mixture of styrene (1.83 mL), acrylic acid (234 mm3), hexadecane (94 mg), divinyl benzene (0.12 g) and compound 3 (0.512 g, 0.358 mmol; 20% of the dry weight; predissolved in 2.0 mL of chloroform) and TOPO (0.208 g, 0.534 mmol) were dissolved in water (40.0 mL) containing sodium dodecyl sulfate (0.09 g) and sodiumborate decahydrate (0.017 g). The resulting suspension was deaerated with argon and homogenized using ultrasound (1 min, 215 W). The resulting emulsion was transferred into a reactor, and it was stirred mechanically (140 rpm) at 60° C. for 20 min under argon (pH 3). The polymerization was initiated by addition of potassium persulphate (0.05 g, predissolved in 3.00 mL of degassed water). The reaction was allowed to proceed for 5 h. The mixture was allowed to cool to RT and purified by dialysis. The following beads were prepared:
10% (w/w) chelate 3 [bead A]
20% (w/w) chelate 3 [bead B]
20% (w/w) chelate 3+1.5 equiv. TOPO, [bead C]
27% (w/w) chelate 9 [bead D]
where % w/w is the of the weight of chelate from the dry weight of the polymerization mixture.
apercentage of dry weight of the chelate in the polymerization mixture;
bcompared to DELFIA enhancement solution;
cmeasured in Tris-saline buffer, pH 7.75;
dTris-saline buffer, pH 7.75 + TOPO + Triton-X.
It will be appreciated that the methods described herein can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.
